Fibroblast growth factor, epidermal growth factor and insulin stimulated phosphorylation of the S6 ribosomal protein in 3T3 cells and ovarian granulosa cells. We are studying the regulation of S6 phosphorylation by these factors in these cells. We have found that there is no correlation between cyclic nucleotide, dependent phosphorylation of S6 observed in vitro and regulation of S6 phosphorylation by peptide growth factors which is observed in the interact cell. We have investigated the effect of mitogenic concentrations of serum on the relative amounts of the two cyclic AMP dependent protein kinase isozymes, types I and II. The ratio of cyclic AMP dependent protein kinase I/II dose not change within the first half hour of mitogen stimulation (when increased S6 phosphorylation occurs), but is changed after four hours of mitogen stimulation. Thus, although the cyclic AMP-dependent protein kinases are regulated by mitogens, the alteration in the relative amount of these two kinases could not be involved in the regulation of S6 phosphorylation. We are currently investigating the contribution of ion fluxes and cytoskeletal components to the regulation of S6 phosphorylation, and are trying to relate, chronologically, the early alterations in transport, phosphorylation and phospholipase activity which occur after stimulating cells with a mitogen.